Báo cáo khoa học: Pulchellin, a highly toxic type 2 ribosome-inactivating protein from Abrus pulchellus Cloning, heterologous expression of A-chain and structural studies ppt
Pulchellin,ahighlytoxictype2 ribosome-inactivating
protein fromAbrus pulchellus
Cloning, heterologousexpressionofA-chainandstructural studies
Andre
´
L. C. Silva
1
, Leandro S. Goto
1
, Anemari R. Dinarte
2
, Daiane Hansen
3
, Renato A. Moreira
4
,
Leila M. Beltramini
1
and Ana P. U. Arau
´
jo
1
1 Centro de Biotecnologia Molecular Estrutural, Instituto de Fı
´
sica de Sa˜o Carlos, Universidade de Sa˜o Paulo, Brazil
2 Fundac¸a˜o Hemocentro de Ribeira˜o Preto, Brazil
3 Universidade Federal de Sa˜o Paulo-EPM, Brazil
4 Universidade Federal do Ceara
´
, Brazil
Ribosome-inactivating proteins (RIPs; EC 3.2.2.22) are
RNA N-glycosidases that depurinate the major ribo-
somal RNA (rRNA), thus damaging ribosomes and
arresting protein synthesis [1]. RIPs are found predom-
inantly in higher plants, but are also present in algae
[2], fungi [3] and bacteria [4]. They vary greatly in their
physical properties and cellular effects [5]. Based on
the structural properties and their corresponding genes,
RIPs have been classified as types 1, 2and 3 [6].
Type 2 RIPs, like ricin and abrin, are highly toxic
heterodimeric proteins that consist ofa polypeptide
with RIP activity (A-chain) linked to a galactose-
binding lectin (B-chain) via a disulfide bond [7]. The
A-chain is the catalytic subunit that exhibits rRNA
N-glycosidase activity by removing a specific adenine
residue froma conserved loop (ricin ⁄ sarcin loop) of
the largest RNA in the ribosome [8]. This modification
induces a conformational change that prevents binding
Keywords
abrin; lectin; ribosome-inactivating protein;
RNA N-glycosidase
Correspondence
A. P. U. Arau
´
jo, Grupo de Biofı
´
sica
Molecular e Espectroscopia, Instituto de
Fı
´
sica de Sa˜o Carlos, Universidade de Sa˜o
Paulo, Caixa Postal 369, CEP 13560-970,
Sa˜o Carlos, SP, Brazil
E-mail: anapaula@if.sc.usp.br
(Received 15 October 2004, revised 6
December 2004, accepted 5 January 2005)
doi:10.1111/j.1742-4658.2005.04545.x
Pulchellin is atype2ribosome-inactivatingprotein isolated from seeds of
the Abruspulchellus tenuiflorus plant. This study aims to obtain active and
homogeneous protein for structuraland biological studies that will clarify
the functional aspects of this toxin. The DNA fragment encoding pulchellin
A-chain was cloned and inserted into pGEX-5X to express the recombinant
pulchellin A-chain (rPAC) as a fusion protein in Escherichia coli. The
deduced amino acid sequence analyses of the rPAC presented a high
sequential identity (> 86%) with the A-chainof abrin-c. The ability of the
rPAC to depurinate rRNA in yeast ribosome was also demonstrated
in vitro. In order to validate the toxic activity we promoted the in vitro
association of the rPAC with the recombinant pulchellin binding chain
(rPBC). Both chains were incubated in the presence ofa reduced ⁄ oxidized
system, yielding an active heterodimer (rPAB). The rPAB showed an
apparent molecular mass of 60 kDa, similar to the native pulchellin. The
toxic activities of the rPAB and native pulchellin were compared by intra-
peritoneal injection of different dilutions into mice. The rPAB was able to
kill 50% of the tested mice with doses of 45 lgÆkg
)1
. Our results indicated
that the heterodimer showed toxic activity anda conformational pattern
similar to pulchellin. In addition, rPAC produced in this heterologous sys-
tem might be useful for the preparation of immunoconjugates with poten-
tial as a therapeutic agent.
Abbreviations
CD, circular dichoism; GST, glutathione S-transferase; LD
50
, median lethal dose; PAC, pulchellin A-chain; RIP, ribosome-inactivating protein;
rPAB, recombinant pulchellin heterodimer; rPAC, recombinant pulchellin A-chain; rPBC, recombinant pulchellin B-chain.
FEBS Journal 272 (2005) 1201–1210 ª 2005 FEBS 1201
of elongation factor 2 (EF2) to the ribosome, resulting
in cell death due to protein synthesis arrest [9]. The
B-chain has lectin properties, preferentially binding to
galactosyl-terminated glycoproteins on the surface of
eukaryotic cells leading to endocytosis. It also facili-
tates A-chain penetration of the lipid bilayer and
entrance into the cytosol [10]. Despite toxic activity,
one group oftype2 RIPs is much less toxic to cells
and animals, but shares structuraland enzymatic prop-
erties with highlytoxic RIPs. This group has been
named nontoxic type2 RIPs [11].
There has been considerable interest in RIPs due to
their potential role in the development of therapeutic
agents. Perhaps the most promising approach to apply-
ing RIPs in therapy is the use of immunotoxins in
which the toxicA-chain is linked to antibodies directed
toward specific cells [12,13]. Several immunotoxins
derived from RIPs have been made and assayed
against specific target cells in vitro and in vivo [14,15].
In addition, RIPs also display antiviral [16], antibacte-
rial [17] and antifungal [18] activities. The apparent
defense role against pathogens also extends to insect
pests [19,20].
Abrus pulchellus tenuiflorus (Leguminosae-Papiliono-
ideae) seeds contain ahighlytoxicprotein named
pulchellin. Pulchellin is atype2 RIP that exhibits
specificity for galactose and galactose-containing struc-
tures, agglutinates human and rabbit erythrocytes, and
kills mice and the microcrustacean Artemia salina at
very low concentrations [21]. Here we report the clo-
ning of pulchellin A-chain (PAC), its cDNA character-
ization, expressionof recombinant toxic A-chain
(rPAC) in Escherichia coli, and the in vitro association
of the rPAC and recombinant pulchellin binding chain
(rPBC) [22], which produces an active heterodimer. We
also performed structuralstudiesof the recombinant
proteins using circular dichroism spectroscopy.
The cloning process will enable the production of
soluble and active homogeneous protein, which is
desirable to the study of its use in immunotherapy.
Comparison of the primary sequences oftype2 RIPs
and their structural characterization will clarify small
differences that significantly change the citotoxity of
such proteins, making them more appropriate for
therapeutic use.
Results
Isolation and cloning of the pulchellin A-chain
gene fragment
Clones of several RIP-2 toxins, such as ricin and abrin
have been obtained in other laboratories and shown to
belong to a multigene family. Also, as with other plant
lectin genes, these genes contain no introns [30–32].
Thus, our initial cloning strategy was based on the
assumption that a similar situation also occurs in pul-
chellin from A. pulchellus based on its phylogenetic
closeness to abrin.
Using degenerated primers, it was possible to
amplify the fragment corresponding to the A-chain
(active) and part of the B-chain (binding). After PCR,
the amplified product was 970 bp, as predicted
based on taxonomic proximity exhibited between pul-
chellin and abrin. The genomic sequence obtained was
submitted to homology search using blast software,
which gave a nucleotide identity of 84% to abrin-c
A-chain precursor fromAbrus precatorius.
Based on the cloned sequence, specific primers were
designed to obtain 5¢-end sequence information via
5¢ RACE. As expected, this amplification product
revealed a band around 450 bp with a high identity to
the preproabrin gene of A. precatorius.
Taken together, the results of genomic cloning and
5¢ RACE, indicated a 34-amino acid N-terminal leader
peptide, 251 residues corresponding to pulchellin
A-chain anda small linker peptide (14 residues) join-
ing the A- and B-chains. As it was found that the
Glu-Asp-Arg-Pro-Ile N-terminal sequence of native
pulchellin A-chain after the amino acid sequence is
very similar to that reported for the N-terminus of the
abrin-c A-chain, it was possible to define the first
amino acid of the mature PAC. Comparing the amino
acid sequences with that of the abrin-c A-chain precur-
sor from A. precatorius , similarities of 88, 86 and 93%,
were found, respectively, for each region. The presence
of both leader and linker peptides, as other type 2
RIPs, is strong evidence that pulchellin is also synthes-
ized as a single chain precursor.
The N-terminal leader sequence directs the immature
precursor to the endoplasmic reticulum [33] and the
linker peptide has been reported as a signal leading the
toxin to the vacuoles [34]. Both the N-terminal leader
and linker peptide are post-translationally excised
resulting in an active toxin comprising two mature
subunits. The overall sequence homology of the pul-
chellin linker peptide is high, differing in only one
amino acid residue among 14 present on the abrin-c
linker, possibly suggesting the same biological roles for
the sequences.
Expression, purification and characterization
of the recombinant pulchellin A-chain
From A. pulchellus genomic DNA, the fragment enco-
ding the mature PAC was amplified by PCR using a
Pulchellin A-chain: cloning andstructuralstudies A. L. C. Silva et al.
1202 FEBS Journal 272 (2005) 1201–1210 ª 2005 FEBS
new set of primers giving rise to a product of
850 bp. The deduced amino acid sequence of this
gene fragment showed a high identity to abrin-c
(86%), abrin-a (78%) and ricin (38%) A-chain
sequences (Fig. 1). The PAC sequence encodes a
mature protein with a predicted molecular mass of
around 29 kDa anda theoretical isoelectric point of
5.5. Alignment of the deduced amino acid sequences
shows that all residues involved in the active site as
described for abrin-a, abrin-c and ricin are conserved
in the sequence reported here. Recent analyses of the
crystal structures of ricin, trichosanthin, pokeweed
antiviral protein, momordin and abrin-a indicate that
the overall architecture of the active site cleft remains
constant in all these proteins [10,35]. In addition, the
sequence of PAC presented only one cysteine residue
that should be involved in the interchain disulfide
bridge.
The DNA fragment encoding PAC was inserted
into a pGEX 5X-1 vector (Amersham-Pharmacia) to
express the recombinant A-chain as aprotein fusion
with glutathione S-transferase (GST). Escherichia coli
AD 202 harboring pGEX-rPAC was used to produce
soluble recombinant fusion protein with the predicted
molecular mass ( 60 kDa) (Fig. 2A). The fusion pro-
tein was purified from the cell lysate by affinity chro-
matography on a glutathione–Sepharose column. After
elution, the fusion protein was submitted to Factor Xa
cleavage for 16 h, at 12 °C. Free recombinant pul-
chellin A-chain (rPAC) was purified in an additional
chromatographic step in a Mono-Q ion-exchange
column. The yield of the rPAC soluble protein was of
3mgÆL
)1
of the Luria–Bertani media culture. The
rPAC was homogeneous upon analysis on 15%
SDS ⁄ PAGE, with an apparent molecular mass of
29 kDa (Fig. 2B). The rPAC was also submitted to
immunodetection using polyclonal antibodies (anti-
native pulchellin), which recognized the recombinant
protein (Fig. 2C).
RNA N-glycosidase activity of the rPAC
An RNA depurination test was used to confirm the
in vitro enzymatic activity of rPAC. Figure 3 shows an
Fig. 1. Deduced amino acid sequence of recombinant pulchellin A-chain (rPAC) aligned to abrin-a, abrin-c and ricin (RTA) A-chains. Conserved
amino acids are highlighted in gray. rPAC residues involved in the potential active site cleft, as predicted by homology to RTA, abrin-a and
abrin-c A-chains, are bold and indicated by *. The cysteine residue (indicated by fl), also due to homology, should be involved in an interchain
disulfide bond.
A. L. C. Silva et al. Pulchellin A-chain: cloning andstructural studies
FEBS Journal 272 (2005) 1201–1210 ª 2005 FEBS 1203
ethidium bromide-stained electrophoresis gel of anil-
ine-treated yeast ribosomal RNA incubated with dif-
ferent amounts of rPAC and native pulchellin (as
positive control). Aniline treatment of rRNA from
yeast ribosomes incubated with RIP at 10, 5 and 1 ng
released a fragment of 370 nucleotides. In contrast,
incubation of ribosome with 0.1 ng did not result in
depurination. The depurination assay performed in the
absence of rPAC or native pulchellin also failed to
generate the RNA fragment. Taken together, these
results suggest that the rPAC possesses RNA N-glyco-
sidase activity just like the native pulchellin.
In vitro association of rPAC and rPBC
In an attempt to check the toxic activity of the rPAC
in vivo, a protocol was used to obtain a functional
heterodimer (named rPAB). The in vitro association of
the two pulchellin subunits (expressed separately) was
achieved by using an oxidized ⁄ reduced system as des-
cribed in Experimental procedures. rPBC, obtained
after the refolding process [22], and rPAC were pooled
and incubated in 50 mm Tris ⁄ HCl buffer 100 mm
NaCl, pH 8.0. Formation of the active rPAB hetero-
dimer could be detected after 2 h incubation (Fig. 4A).
At 4 °C, a plateau of recombinant heterodimer forma-
tion was reached 48 h after the onset of the experi-
ment. The yield of the rPAB association process was
0.2 mg, corresponding to 20% of the total theoretically
obtainable heterodimer. After association, the protein
was loaded into a CentriPrep (30 000 cut-off, Milli-
pore) and dialofiltrated against the incubation buffer
to separate the heterodimer from free rPAC and rPBC.
Figure 4(B) shows the purity of the rPAB heterodimer
after dialofiltration, under reducing (lane 1) and non-
reducing (lane 2) conditions in SDS ⁄ PAGE silver-
stained. An apparent molecular mass of 59 kDa for
ABC
Fig. 2. (A) A-chainexpression analysis in SDS ⁄ PAGE, 15%. Lane 1, molecular mass marker; lanes 2and 3, total proteins from E. coli
AD 202–pGEX-rPAC not induced and induced by 0.4 mM isopropyl thio-b-D-galactopyranoside, respectively; lane 4, soluble fraction from cellu-
lar lysates after sonication; lane 5, insoluble fraction; lane 6, fusion protein (A-chain plus GST) eluted from affinity resin. (B) rPAC purification
analysis in SDS ⁄ PAGE, 15%. Lane 1, molecular mass marker; lane 2, fusion protein (GST + PAC) after Factor Xa cleavage; lane 3, samples
eluted from the major peak of the Mono-Q, corresponding to the rPAC; lane 4, fraction corresponding to GST. (C) Western blot analysis
using rabbit polyclonal antibodies against native pulchellin. Lane 1, rPAC; lane 2, native pulchellin.
Fig. 3. N-glycosidase activities of rPAC and native pulchellin. Yeast
ribosomes (20 lg) were incubated with different amounts (10, 5, 1
and 0.1 ng) of rPAC and native pulchellin for 1 h at 25 °C. The
rRNAs were extracted and treated with 1
M aniline-acetic for 4 min
at 60 °C. Samples were analyzed by denaturing agarose–formamide
gel electrophoresis and staining with ethidium bromide. Yeast ribo-
somes samples treated with rPAC (lanes 1–4), native pulchellin
(lanes 5–8) and without treatment (negative control) (lanes 9–10)
are shown. The arrow indicates the position of the small RNA frag-
ment released upon aniline treatment of rRNA. +, presence of anil-
ine treatment; –, absence of aniline treatment.
Pulchellin A-chain: cloning andstructuralstudies A. L. C. Silva et al.
1204 FEBS Journal 272 (2005) 1201–1210 ª 2005 FEBS
the heterodimer is expected because the molecular mas-
ses of rPAC and rPBC, are 29.2 and 29.8 kDa [22],
respectively. The native pulchellin has an apparent
molecular mass of 60 kDa [21] due to the native glyco-
sylation process [36].
Circular dichroism and biological activity
of the rPAB heterodimer
Circular dichroism (CD) measurements and biological
tests were used to investigate the similarity between the
native pulchellin and the rPAB heterodimer. Figure 5
shows the far-UV CD spectra of rPAC, rPBC, rPAB
and native pulchellin. CD analyses for the rPAC
sample showed aprotein profile with predominance of
a-helical elements [37]: two negative bands at 222 and
208 nm anda positive peak at 196 nm. The CD spec-
trum shape of refolded rPBC showed one maximum
band at 231 nm, two minima at 214 and 206 nm, and
a negative to positive crossover at 199 nm. This spec-
trum showed that the b-sheet was the predominant
component present in rPBC secondary structure. When
compared, both native pulchellin and rPAB hetero-
dimer presented very similar CD spectra.
The biological activity of the rPAB heterodimer in
terms of lethal dose (LD
50
) values is given in Fig. 6.
After 48 h, the rPAB was able to kill 50% of mice tes-
ted with a dose of 45 lgÆkg
)1
, which was a little less
toxic than the lethal dose found for native pulchellin
AB
Fig. 4. In vitro association of rPAC with
rPBC. (A) rPAC was incubated with rPBC in
the presence ofa reduced ⁄ oxidized cysteine
system at 4 °C for 48 h. The reaction prod-
ucts were analyzed using 15% SDS ⁄ PAGE
and were silver-stained. Lane M, protein
marker; numbered lanes correspond to
incubation times. rPAB heterodimer appears
as an additional band of 60 kDa after 2 h
incubation (lanes 2–48). (B) rPAB hetero-
dimer after dialofiltration, under reducing
(lane 1) and nonreducing (lane 2) conditions.
Fig. 5. CD spectra of recombinant pulchellin A-chain (rPAC), recom-
binant pulchellin B-chain (rPBC), recombinant pulchellin (rPAB) and
native pulchellin. Spectra were obtained from each protein at a con-
centration of 0.3 mgÆmL
)1
in 20 mM Tris ⁄ HCl, pH 8.0. Measure-
ments were performed using quartz cuvettes of 1 mm path length
and recorded from 195 to 250 nm as the average of 16 scans at
25 °C.
buffer
100
90
80
70
60
50
Death (%)
40
30
20
10
0
15
30
Dose (µ
g.Kg
–1
animal)
45
50
60
Pulchellin
rPAB
rPAC
rPBC
Fig. 6. Lethal activity determined by intraperitoneal injection in mice
using different concentrations of recombinant pulchellin A-chain
(rPAC), recombinant pulchellin B-chain (rPBC), recombinant pulchel-
lin (rPAB) and native pulchellin (as positive control). The buffers of
each protein were used as negative controls. Groups of six animals
and different doses of each protein were prepared. Each group rep-
resented a dose and the toxic effects were determined after 48 h.
A. L. C. Silva et al. Pulchellin A-chain: cloning andstructural studies
FEBS Journal 272 (2005) 1201–1210 ª 2005 FEBS 1205
(30 lgÆkg
)1
). Sublethal doses also lead to animal death
some days later until the end of experiments. Although
this value is higher than found for other similar toxins
[38], the toxic effects observed agree with those
induced by type2 RIPs. The structuraland biological
properties determined for the rPAB heterodimer
showed that this protein presents similar behavior to
that of the native pulchellin.
Discussion
Pulchellin, atype2 RIP isolated from A. pulchellus
seed, is a potent plant toxin, similar to abrin and ricin.
Cloning of the coding gene from pulchellin A-chain
will greatly facilitate the understanding of the protein
structure and function, and lay a solid foundation for
its application. This study reports the cloning and
characterization of the A-chain gene that encodes the
toxic chain of pulchellin.
rPAC was expressed in a soluble form, preserving its
structure and biological activity. Its DNA sequence
has very high identity with (93.0%) anda similar size
to (251 bp) abrin-c A-chain [39]. The molecular mass
of rPAC (29 kDa) is consistent with that reported for
native pulchellin A-chain [21]. rPAC was found to be
highly homologous to other type2 RIPs [30,40]. As
shown in Fig. 2, rPAC shows a high sequence homo-
logy to A-chains from abrins. In the four RIPs listed,
there is one conserved cysteine residue close to the
C-terminal of the A-chains, which allows formation of
one interchain bond with another conserved cysteine
residue in their respective B-chains. The active RNA
N-glycosidase sites of abrin-a, abrin-c and ricin are
composed of five invariant residues (Tyr74, Tyr113,
Glu164, Arg167 and Trp198 in abrin-a and abrin-c,
and Tyr115, Tyr158, Glu212, Arg215 and Trp246 in
ricin) and another five conserved residues (Asn72,
Arg124, Gln160, Glu195 and Asn196 in abrin-a and
abrin-c and Asn78, Arg134, Gln172, Glu208 and
Asn209 in ricin) [30,35]. The alignment of amino acid
sequences shows that all residues involved in the active
site cleft of abrin-a, abrin-c and ricin are totally con-
served in the rPAC sequence.
The N-glycosidade activity assays showed that rPAC
was enzymatically active. RIP-mediated depurination
of the large ribosomal subunit RNA results in a sus-
ceptibility of the RNA sugar–phosphate backbone to
hydrolysis at the depurination site, which leads to the
release ofa small fragment of 130–400 nucleotides
from the 3¢-end of the rRNA [41,42]. This fragment is
diagnostic of RIP-catalyzed depurination and is readily
observed following agarose–formamide gel electro-
phoresis [43]. rPAC (1 ng) was able to cleave the
N-glycosidic bond of yeast rRNA, releasing an RNA
fragment of 370 nucleotides after treatment with
aniline, as did native pulchellin. Thus, this activity can
be attributed to conserved residues that form the active
site of RNA N-glycosidase in rPAC. Stirpe et al. [44]
showed that a fragment of 400 nucleotides arises
from removal of A3024 in yeast 26S rRNA when incu-
bated with ricin.
Using the intraperitoneal toxicity test to compare
the potency and activity of rPAB heterodimer and
native pulchellin, no significant differences between the
recombinant heterodimer and native protein were
found. Neither rPAC nor rPBC had atoxic effect on
mice in the dosage range used. Thus, it is clear that
in vivo poisoning occurs only if the whole heterodi-
meric protein (rPAB) is administered. This activity was
expected because the CD results show that rPAB has
the same CD profile and consequently, has a secon-
dary structure fold similar to the native pulchellin. Our
results are in accordance those of with Eck et al. [45],
who compared the toxic activities of single chains from
plant mistletoe lectin (pML) with the recombinant
mistletoe lectin heterodimer (rML), concluding that
both lectin and rRNA N-glycosidase activities are pre-
requisites for cytotoxic effect on target cells.
In addition, our results also suggest that glycosyla-
tion is not essential for heterodimer internalization
because the rPAB heterodimer is derived from biosyn-
thesis in E. coli (therefore it is not glycosylated) and
showed toxicity similar to that of native pulchellin.
In fact, the absence of glycolysation has advantages
when using the A-chains in immunotoxins. For exam-
ple, deglycosylated ricin A-chain (dgA) immunotoxins
greatly reduced the levels of nonspecific uptake by the
liver and concomitantly increased tumor-specific local-
ization [46,47].
Regarding the therapeutic use of immunotoxins,
an important consideration for immunoconjugate
assembly is the nature of the linkage between anti-
body and RIP [47]. A disulfide bridge is usually
thought to be essential for maximal cytotoxicity.
Most type 1 RIPs do not have any free cysteine resi-
dues [48], which implies the need for modification of
both antibody and RIP with chemical agents to pro-
duce the disulfide bond. Fortunately, rPAC has one
free cysteine located in the C-terminal region and
can directly form a disulfide bond with an activated
antibody thiol group via a disulfide-exchange reac-
tion. Therefore, rPAC is easily produced in a
heterologous system and it might be useful for the
preparation of immunoconjugates with great poten-
tial as a chemotherapeutic agent for the treatment of
cancer [11,47,49] and AIDS [50,51].
Pulchellin A-chain: cloning andstructuralstudies A. L. C. Silva et al.
1206 FEBS Journal 272 (2005) 1201–1210 ª 2005 FEBS
Experimental procedures
Materials
E. coli DH5-a (Promega, Madison, WI, USA) was used for
plasmid amplification and E. coli ad 202 strain (Novagen,
Madison, WI, USA) was used to express the gene.
pGEX 5X-1 expression vector was purchased from Amer-
sham-Pharmacia Biotech (Piscataway, NJ, USA). Isopropyl
thio-b-d-galactoside was purchased from Sigma (St. Louis,
MO, USA). Oligonucleotide synthesis was produced by
Gibco BRL (Rockville, MD, USA). Restriction endonuc-
leases, and DNA ladders were obtained from Promega.
Factor Xa protease was purchased from Biolabs (Beverly,
MA, USA). All other chemicals used were analytical grade.
Plant material and nucleic acid isolation from
A. pulchellus
A. pulchellus tenuiflorus subspecies were cultivated in our
laboratories to produce the necessary tissues for nucleic
acid extractions. Approximately 1.5 g of leaves were frozen
and ground to powder in liquid nitrogen. Genomic DNA
was further isolated using a plant genomic DNA isolation
Floraclean kit (Qiagen, Valencia, CA, USA), following the
manufacturer’s instructions.
Total RNA was isolated from immature A. pulchellus
seeds, previously frozen in liquid nitrogen, using the RNA-
easy Plant Mini Kit (Qiagen). The total RNA was quanti-
fied at 260 nm (Hitachi U-2000 spectrophotometer) and
2 lg was used to 5¢RACE.
Genomic cloning
Degenerate primers were designed based on the amino acid
sequence conservations along the preproabrin gene (MED-
LINE 91266957) and were used for genomic amplifications.
Their design was based on the A. precatorius codon table,
trying to minimize the degeneration at their 3¢ ending. A
pair of degenerate primers (abrin 1: 5¢-ACTGAAGGTGCC
ACTTCACAAAGCTAYAARCARTT-3¢; abrin 3: 5¢-GGT
TAAACACTTCCCGTTGGACCTDATNGT-3¢) was cho-
sen to represent the possible coding sequences of the con-
served N-terminus of the pulchellin A- and B-chains. Thus,
the expected amplified product could represent the major
sequence encoding the A-chainand an additional fragment
encoding part of the B-chain.
The primers described above were used in a PCR con-
taining the A. pulchellus genomic DNA as a template. The
reaction mixture included: 100 pmol of each primer; 1.0 lg
of A. pulchellus DNA template; 200 lm for each dNTP; 1·
PCR buffer (Amersham-Pharmacia Biotech); 2.5 U Taq
DNA polymerase (Amersham-Pharmacia Biotech) in a total
volume of 50 lL. PCR was performed for: 1 cycle at 94 °C
for 5 min; 30 cycles at 94 °C for 1 min, 45 °C for 1 min,
and a primer extension for 1 min at 72 °C; anda final cycle
at 72 ° C for 7 min. The products obtained by amplification
were cloned in the pGEM-T easy vector (Promega), which
was used to transform E. coli DH5-a competent cells.
Sequencing
The positive clones were sequenced in the ABI-Prism 377
(Perkin–Elmer) automatic sequencer following the manufac-
turer’s instructions. The whole fragment was sequenced and
submitted to a blast script data bank search [23].
RACE
The 5¢ RACE was performed using Access RT-PCR
Introductory System according to an adapted protocol
previously described [24]. Terminal transferase (Life
Technologies, Rockville, MD, USA) was used to add a
homopolymer G-tail in the first strand for 5¢ RACE. Speci-
fic primers were designed for this step based on DNA
sequences obtained previously. Thus, the sequences of the
primers used for 5¢ RACE were: 5¢-GGGCATCACGGA
AGAAATAG-3¢ for a reverse transcription and 5¢-GC
TCTAGAGCATTCGTCACATCGATACC-3¢ with 5¢-AA
GGAATT(dC)14 for the following amplification. The ther-
mal profile was 40 cycles of 96 °C for 1 min, 55 °C for
2 min, 72 °C for 3 min anda final extension for 10 min at
72 °C. The PCR products were analyzed on 1% agarose
gels. Subsequently, the RACE reaction product was puri-
fied and inserted into vector pGEM-T (Promega). One
microliter of this mixture was used to transform E. coli
EletroMax DH5a-E cells (Gibco-BRL) by electroporation.
The positive clones were sequenced was already described.
Pulchellin A-chain cloning and expression
A new oligonucleotide set was then synthesized to amplify
the pulchellin A-chain gene fragment from A. pulchellus
(GenBank accession number AY781337) by PCR. The
sequences of the synthetic oligonucleotides used for amplifi-
cation were pulcA ⁄ BamHI (5¢-CG
GGATCCAGGAGGAC
CGGCCCATTGAATTTACTACTG-3¢, the BamHI restric-
tion site included is underlined) and the reverse primer
pulcA ⁄ NotI(5¢-ATAGTTTA
GCGGCCGCTCAATTTGGC
GGATTGCAGAC-3¢, NotI restriction site is underlined).
The product obtained by amplification was inserted into
pGEX 5X-1 (Amersham-Pharmacia Biotech). Briefly,
amplification was carried out in a 50 lL reaction volume
containing 625 ng genomic DNA, 100 pmol of each pri-
mer, 0.2 mm dNTPs and2 U of Deep Vent DNA poly-
merase (Biolabs) in the PCR buffer recommended by the
enzyme manufacturer. Cycling parameters were: 1 cycle at
96 °C for 5 min, 5 cycles (94 °C for 1 min, 57 °C for
A. L. C. Silva et al. Pulchellin A-chain: cloning andstructural studies
FEBS Journal 272 (2005) 1201–1210 ª 2005 FEBS 1207
1.5 min and 72 °C for 1 min), 25 cycles (94 °C for 1 min,
60 °C for 1.5 min and 72 °C for 1 min) followed by 10 min
at 72 °C to a final extension. Both amplified fragment and
pGEX 5X-1 vector were digested with BamHI and NotI
endonucleases and purified. Such digestion resulted in cohe-
sive sticky ends able to directionally insert ligation, which
was performed by a T4 DNA ligase reaction. E. coli
DH5-a competent CaCl
2
cells were transformed with the
recombinant plasmid (named pGEX-rPAC) by heat shock
treatment [25].
The expression plasmid pGEX-rPAC was used to trans-
form competent E. coli ad 202 strain. The transformed cells
ad 202 pGEX-rPAC were grown at 37 °C in Luria–Bertani
medium supplemented with kanamycin (50 lgÆmL
)1
) and
cultured up to a cell density absorbance of A
600
¼ 0.4–0.6.
Once this density was reached, the expressionof recombin-
ant protein was induced with 0.4 mm isopropyl thio-
b-d-galactopyranoside and carried out for 12 h at 20 °C.
Before and after induction, cell aliquots were collected by
centrifugation and analyzed by 15% SDS ⁄ PAGE [26]. The
remaining cells were pelleted by centrifugation and resus-
pended in 8 mL of 0.1 m pH 8.0 NaCl ⁄ P
i
buffer containing
1.0 mgÆmL
)1
lysozyme. After 30 min incubation on ice,
cells were disrupted by sonication and the lysate was clar-
ified by centrifugation at 20 000 g. At this point, both pellet
and supernatant were submitted to SDS ⁄ PAGE 15% to
check the solubility of the recombinant pulchellin A-chain
(named rPAC). The clear supernatant was used for the
purification step.
Purification of rPAC
The supernatant obtained was applied to a2 mL glutathi-
one–Sepharose 4 Fast Flow (Amersham-Pharmacia) and
the column was washed with 10 vol. of NaCl ⁄ P
i
. After this,
5 vol. of the elution buffer (50 mm de Tris ⁄ HCl, 10 mm of
reduced glutathione, pH 8.0) were loaded and the recom-
binant A-chain was collected. This recombinant protein
was eluted, pooled and submitted to Fator Xa cleavage
protocol followed by an additional chromatographic step in
the Mono-Q HR 5 ⁄ 5 (1 mL).
Western blot analysis rPAC was submitted at immuno-
detection, after SDS ⁄ PAGE, onto nitrocellulose membranes
(Protan, Keene, NH, USA), using a Bio-Rad electrotransfer
cell, for 2 h at 110 V. Membranes were developed with a
secondary antibody–alkaline phosphatase detection system
(Promega), using rabbit polyclonal antibodies produced
against native pulchellin. An antiserum titer of 1 : 5000 was
used for all experiments.
Assay of the N-glycosidase activity of rPAC
The isolation of yeast (Pichia pastoris) ribosome was per-
formed as previously described [27]. Yeast ribosomes
(20 lg) were incubated at 25 °C for 1 h with different
amounts of rPAC (0.1, 1, 5 and 10 ng) in buffer A (20 mm
Tris ⁄ HCl pH 8.0, 100 mm NaCl) in a total volume of
20 lL. The reaction was stopped by the addition of 0.1%
SDS. The rRNA was obtained by phenol–chloroform
extraction and precipitated by the addition of 0.1 vol. 2 m
NaOAc pH 6.0 and 2.5 vol. 100% ethanol. The reaction
mixtures were frozen and the precipitated rRNA was
pelleted by centrifugation at 13 000 g for 30 min at 4 °C.
The pellets were washed once with 70% ethanol and dried
for 20 min in a vacuum desiccator. rRNA (10 lg) was
treated (for 4 min, at 60 °C) with 20 lLof1m aniline-acetic
(pH 4.5) or 20 lLofH
2
O for nonaniline-treated controls.
The reactions were stopped by the addition of 0.1 vol. of
NH
4
OAc and 2.5 vol. of 100% ethanol and frozen before
centrifugation for 1 h at 4 °C. The pellets were resuspended
in 15 lL of 60% formamide ⁄ 0.1· TPE (3.6 mm Tris, 3 mm
NaH
2
PO
4
, 0.2 mm EDTA) mix and run on a denaturing
agarose–formamide gel electrophoresis. The RNA was
visualized on a short-wave ultra-violet transilluminator.
In vitro association of rPAC and rPBC
The recombinant pulchellin heterodimer (named rPAB) was
prepared by coupling isolated, rPAC and the recombinat
pulchellin binding chain (rPBC). The rPBC was produced
as described previously by Goto et al. [22].
For association of rPAC and rPBC, the two chains
(0.5 mg of each chain) were incubated in 50 mm Tris ⁄ HCl
buffer, 100 mm NaCl, pH 8.0 at 4 °C for 48 h. For the
formation of interchain disulfide bridges, the reaction was
incubated in the presence ofa reduced ⁄ oxidized system
(cysteine to cystine ratio 5 : 1). The association process was
followed by 15% SDS ⁄ PAGE under nonreducing condi-
tions. Silver staining was performed as described by Blum
et al. [28].
Circular dichroism measurements
CD spectra were recorded with a Jasco J-715 spectropola-
rimeter over a wavelength range of 195–250 nm. Measure-
ments were made in quartz cuvettes of 1 mm path length,
recorded as an average of 32 scans. CD spectra were meas-
ured in protein solutions of 0.3 mgÆmL
)1
. CD spectra were
obtained in millidegrees and converted to molar ellipticity.
Secondary structure fractions were calculated from decon-
volution of the CD spectra using the program selcon 3
[29] employing a database of 42 proteins.
Biological activity in vivo of the rPAB
The biological activity of the recombinant pulchellin was
studied by measuring its toxic activity (in vivo). Toxic activ-
ity was determined by intraperitoneal injection in mice
using different doses (15, 30, 45, 50 and 60 l g Æ kg
)1
of
Pulchellin A-chain: cloning andstructuralstudies A. L. C. Silva et al.
1208 FEBS Journal 272 (2005) 1201–1210 ª 2005 FEBS
animal body mass) of recombinant pulchellin. Native pul-
chellin, produced as described by Ramos et al. [21], rPAC
and rPBC were used as controls. Groups of six animals and
different doses of each protein were prepared. Each group
represented a particular dose and each animal in the same
group received the same dose ofprotein in proportion to
their body mass. After injection of each dose, the toxic
effects were determined after 48 h and acute LD
50
values
were calculated.
Acknowledgements
We thank Dr Heloı
´
sa S. S. de Arau´ jo for N-terminal
analysis, and Andressa P. A. Pinto for contributions to
this study. This work was supported by grants from
the Conselho Nacional de Desenvolvimento Cientı
´
fico
e Tecnolo
´
gico (CNPq) and Fundac¸ a
˜
o de Amparo a
`
Pesquisa do Estado de Sa
˜
o Paulo (FAPESP).
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Cloning, heterologous expression of A- chain and structural studies
Andre
´
L degeneration at their 3¢ ending. A
pair of degenerate primers (abrin 1: 5¢-ACTGAAGGTGCC
ACTTCACAAAGCTAYAARCARTT-3¢; abrin 3: 5¢-GGT
TAAACACTTCCCGTTGGACCTDATNGT-3¢)